Regulated Osteochondrogenesis of Human Mesenchymal Stem Cells Using Gene Delivery

描述（由申请人提供）：尽管进行了广泛的骨科研究，但因疾病或损伤而受损的关节软骨的再生是一个复杂的问题，仍然是一个重大的临床挑战。虽然有许多止痛药，治疗策略和外科手术的发展，以解决这一健康问题，大多数治疗是短暂的和非补救性的。该项目的全球目标是用一种多层结构修复骨软骨缺损，该结构具有空间控制的成软骨和成骨特性，并由人间充质干细胞（hMSCs）产生。为此，提出了以下具体目标：（1）开发3D控释系统，其以适合于MSC的成骨和成软骨分化的速率和浓度递送质粒编码的转录因子，和（2）评价在具体目标1中创建的控释系统产生双层成软骨和成骨构建体的潜力。MSC在3D支架中的骨软骨细胞分化将通过用于软骨形成的转录因子Sox-5、Sox-6、Sox-9（Sox trio）和用于骨形成的Runx-2的基因递送来实现。这些质粒中的每一个都将与我们实验室开发的新型聚合基因递送载体（支化聚乙烯亚胺和透明质酸的缀合物）复合以增加转染效率。最初，将检查质粒浓度和暴露持续时间对骨软骨细胞分化的影响，以确定适当的靶向释放曲线。然后将用包埋在聚合物纤维的芯中的载体-质粒复合物制造电纺的同轴纤维网支架，并且将利用良好建立的制造变量来实现所需的释放曲线。对设想的最终双层结构的各个层的评价将基于接种的MSC的成骨和成软骨分化以及类似于天然组织的细胞外基质的产生。这些研究将涉及hMSCs，以评估所提出的方法在人类中最终临床应用的初步可行性，同时将使用兔MSCs进行平行研究，以评估兔动物模型在这种骨软骨组织再生新方法的转化临床前开发中的预期应用的适当性。该提议的新奇在于其利用（a）MSC来规避与分化细胞类型的有限可用性和维持相关的传统问题，（B）质粒中编码的转录因子来提供MSC的更广泛的表型诱导，以及（c）聚合物支架来提供用于细胞增殖/分化的三维支撑网格和基因递送载体的受控释放。虽然该提案解决了特定的临床需求，但这些研究的影响并不限于软骨甚至骨科应用。本文提出的原理提供了一种避免与分化细胞的分离和维持相关的问题的方法。转录因子将潜在地促进hMSCs在许多组织工程应用中的使用。此外，新的支架制造技术允许多种基因递送载体的调节递送，同时为细胞生长提供3D支持。这些技术在组织工程中具有广泛的应用，并且可以潜在地用于创建模仿软骨和其他复杂组织的带状结构的多层支架。 公共卫生相关性：该项目提出了一种创新的技术和方法，用于创建骨软骨结构，用于修复受损的关节软骨和软骨下骨。将在新型控释系统中利用发育生物信号的组合来递送差异信号以诱导人类间充质干细胞进入适当的骨软骨表型。该系统将增加组织工程师可用于生成具有多种细胞类型的各向异性组织的技术范围。

DESCRIPTION (provided by applicant): Regeneration of articular cartilage damaged either by disease or injury is a complex problem that remains a significant clinical challenge despite extensive orthopaedic research. Although there are numerous analgesics, therapeutic strategies and surgical procedures developed to address this health concern, most of the therapies are short-lived and non-remedial. The global objective of this project is to repair osteochondral defects with a multilayered construct presenting spatially controlled chondrogenic and osteogenic properties and generated from human mesenchymal stem cells (hMSCs). To this end, the following specific aims are proposed: (1) to develop 3D controlled release systems that deliver plasmid encoded transcription factors at rates and concentrations appropriate for osteogenic and chondrogenic differentiation of MSCs and (2) to evaluate the controlled release systems created in Specific Aim 1 for their potential to generate bi-layered chondrogenic and osteogenic constructs. Osteochondral differentiation of MSCs in 3D scaffolds will be achieved through gene delivery of transcription factors Sox-5, Sox-6, Sox-9 (Sox trio) for chondrogenesis, and Runx-2 for osteogenesis. Each of these plasmids will be complexed with a novel polymeric gene delivery vector developed in our laboratory (a conjugate of branched polyethylenimine and hyaluronic acid) to increase transfection efficiency. Initially, the effect of plasmid concentration and exposure duration on osteochondral differentiation will be examined to identify the appropriate target release profiles. Electrospun, co-axial fiber mesh scaffolds will then be fabricated with the vector-plasmid complexes embedded in the core of the polymer fibers and well-established fabrication variables will be utilized to achieve the desired release profiles. Evaluation of the individual layers for the envisioned final bi-layered construct will be based upon the osteogenic and chondrogenic differentiation of the seeded MSCs and the generation of extracellular matrix similar to the native tissue. The studies will involve hMSCs to evaluate the initial feasibility of the proposed approach for ultimate clinical application in humans, while parallel studies will be conducted using rabbit MSCs to evaluate the appropriateness of the envisioned application of the rabbit animal model in the translational pre-clinical development of this novel approach to osteochondral tissue regeneration. The novelty of this proposal is that it utilizes (a) MSCs to circumvent the traditional problems associated with the limited availability and maintenance of differentiated cell types, (b) transcription factors encoded in plasmids to provide a broader phenotypic induction of MSCs and (c) polymeric scaffolds to provide a three dimensional support lattice for cell proliferation/differentiaton and the controlled release of gene delivery vectors. Although this proposal addresses a specific clinical need, the impact of these studies is not limited to cartilage or even orthopaedic applications. The principles proposed herein provide a means to circumvent the problems associated with the isolation and maintenance of differentiated cells. Transcription factors will potentially facilitate the use of hMSCs in a number of tissue engineering applications. Furthermore, the novel scaffold fabrication technique permits regulated delivery of multiple gene delivery vectors while providing a 3D support for cell growth. These techniques have wide-ranging applications in tissue engineering and can potentially be used to create multilayer scaffolds that mimic the zonal architecture of cartilage and other complex tissues. PUBLIC HEALTH RELEVANCE: This project proposes an innovative technology and approach for creating osteochondral constructs for the repair of damaged articular cartilage and subchondral bone. A combination of developmental biological signals will be utilized in a novel controlled release system to deliver differential signals to induce human mesenchymal stem cells into appropriate osteochondral phenotypes. This system will increase the scope of techniques available to tissue engineers for generating anisotropic tissues with multiple cell types.